ALKBH5去除NFE2L1的m6A甲基化修饰促进黑色素瘤放化疗抵抗的机制研究

ALKBH5 demethylates m6A modifications in NFE2L1 and induces resistance to chemoradiotherapy of cutaneous malignant melanoma

目的探讨去甲基化酶AlkB同源蛋白5(AlkB homologue 5,ALKBH5)介导的m6A甲基化修饰在皮肤恶性黑色素瘤(cutaneous malignant melanoma,CMM)放化疗抵抗中的作用。方法自2019年5月至2021年1月,辽宁省人民医院皮肤科采用实时荧光聚合酶链反应(real-time polymerase chain reaction,RT-PCR)检测CMM细胞中ALKBH5和NFE2L1(nuclear factor,erythroid 2 like 1)的表达。TCGA分析ALKBH5和NFE2L1的表达及相关性。在CMM细胞中敲减ALKBH5,免疫沉淀检测NFE2L1的m6A甲基化表达变化。MTT检测ALKBH5和NFE2L1在CMM细胞放化疗抵抗中的作用。结果与正常人表皮黑色素细胞相比,CMM细胞中ALKBH5和NFE2L1的表达水平均明显升高(P<0.05);ALKBH5调控NFE2L1的m RNA的m6A甲基化水平。敲减ALKBH5的CMM细胞,在经过照射和顺铂处理后,细胞成活比例下降,放化疗抵抗减轻(P<0.05);而过表达NFE2L1,治疗后的细胞成活比例增加,诱导了CMM的放化疗抵抗(P<0.05)。结论ALKBH5可通过去除NFE2L1的m6A甲基化修饰,上调NFE2L1的表达,促进CMM的放化疗抵抗。ALKBH5对CMM恶性表型的影响是通过NFE2L1实现的。

Objective To explore the role of AlkB homologue 5(ALKBH5)mediated m6A methylation modifications in the chemoradiotherapy resistance of cutaneous malignant melanoma(CMM).Methods From May 2019 to January 2021,the expression of ALKBH5 and NFE2L1(nuclear factor,erythroid 2 like 1)in CMM cells was detected by real-time polymerase chain reaction(RT-PCR).TCGA was used to analyze the expression and correlation of ALKBH5 and NFE2L1.ALKBH5 was knocked down in CMM cells and the changes in m6A methylation expression of NFE2L1 was detected by immunoprecipitation.The role of ALKBH5 and NFE2L1 in chemoradiotherapy resistance of CMM cells was detected by MTT.Results The expression of both ALKBH5 and NFE2L1 in CMM cells was significantly higher than that in normal human epidermal melanocytes(P<0.05).The m6A methylation level of NFE2L1 mRNA was regulated by ALKBH5.In CMM cells with ALKBH5 knocked down,the survival rate of cells decreased and the chemoradiotherapy resistance reduced after irradiation and cisplatin treatment(P<0.05).After overexpression of NFE2L1,the survival rate of cells increased after treatment and the chemoradiotherapy resistance of CMM was induced(P<0.05).Conclusion ALKBH5 promoted chemoradiotherapy resistance of CMM by removing the m6A methylation modification of NFE2L1 and upregulating NFE2L1 expression.The effect of ALKBH5 on malignant phenotype of CMM was mediated through NFE2L1.